RESUMO
Genetic alteration of the RET proto-oncogene is associated with multiple endocrine neoplasia type 2A and 2B (MEN 2A and MEN 2B), familial medullary thyroid carcinoma (FMTC) and Hirschprung's disease. Oncogenically activated RET has also been demonstrated in sporadic medullary thyroid tumors, which in some cases show somatic missense mutations. We have recently described a complex 9 bp deletion in RET exon 11 in a single case of sporadic MTC. In order to determine the prevalence of this mutation among sporadic MTC tumors, we have now analysed 15 cases and five normal controls by PCR-based nonradioactive single-strand conformational polymorphism analysis (PCR-SSCP) and fragment size analysis of exon 11. DNA was extracted from microdissected tumor tissue or normal cells and subjected to nested PCR prior to analysis. A markedly divergent SSCP pattern and a PCR fragment 9 bp shorter than normal were demonstrated in 14 of the 15 MTC tumors. Sequencing revealed the deletion of nine bases encompassing a key cysteine at codon 634, often altered in MEN 2A. Four lymphocyte controls and normal thyroid tissue from one patient failed to show the deletion. Several factors in the DNA sequence environment immediately surrounding the deletions, including an extended inverted repeat, several direct repeats and a so-called symmetric element suggest that the deletional events may be non-random.
Assuntos
Carcinoma Medular/genética , Proteínas de Drosophila , Éxons/genética , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/genética , Receptores Proteína Tirosina Quinases/genética , Deleção de Sequência , Neoplasias da Glândula Tireoide/genética , Sequência de Bases , Códon/genética , Análise Mutacional de DNA , DNA de Neoplasias/genética , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-retRESUMO
Over the past decade molecular genetic methods have played an increasingly important role in the diagnosis of hematologic malignancies. Moreover, they have provided a tool to analyze many of the non-random cytogenetic anomalies associated with hematologic neoplasias, contributing considerably to our understanding of several of those diseases, and to improving diagnostic accuracy. The rapid development of molecular genetics progressively allows the replacement of time-consuming and technically demanding procedures. Even more relevant are the new clinical applications that already include the search for valuable prognostic information and ways of evaluating minimal residual disease or recognizing early relapsing disease. This paper is a critical but necessarily simplified overview of the main contributions of molecular genetics to the field of hematopathology. We discuss the information provided by several molecular methods within different clinical contexts, covering common problems in diagnostic pathology as well as prognostic evaluation and therapy monitoring.
Assuntos
Leucemia/diagnóstico , Linfoma/diagnóstico , Aberrações Cromossômicas/diagnóstico , Transtornos Cromossômicos , DNA de Neoplasias/genética , Genes de Imunoglobulinas , Genes Codificadores dos Receptores de Linfócitos T , Humanos , Imunofenotipagem , Neoplasia Residual/diagnóstico , Prognóstico , RecidivaRESUMO
The polymerase chain reaction (PCR), used to detect human papillomavirus (HPV), is finding increasing applications in clinical laboratories. The standard method of analysis to detect amplified PCR products is ethidium bromide gel electrophoresis combined with labor intensive blot hybridization. In this study, we describe single-strand conformation polymorphism (SSCP) to detect and genotype simultaneously general primer GP5+/GP6+ amplified HPV DNA using semiautomated electrophoresis on polyacrylamide gels (PAGE) combined with sensitive silver staining. To establish a standard for the band patterns of the various HPV types, we used HPV plasmid DNA, which allowed us to distinguish HPV 6, 11, 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58, covering the most frequently recognized types. All the types tested are separated from each other, demonstrating diverse band patterns, HPV 16 being the most distinct. We also investigated PCR-SSCP for HPV detection and typing of 86 cervical biopsies diagnosed as cervical intraepithelial neoplasia (CIN) I-III and known to be HPV positive by PCR-slot blot hybridization and in situ hybridization. The correlation with SSCP was 91% for in situ hybridization and 98% for PCR-slot blot hybridization. SSCP is reproducible and specific. Its sensitivity is comparable to slot-blot hybridization. The interval to SSCP is approximately 2 h after PCR compared with several days' work when using conventional blot hybridization. We concluded that SSCP may be more advantageous than other PCR-based typing technologies.
Assuntos
Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/genética , Polimorfismo Conformacional de Fita Simples , Adolescente , Adulto , Feminino , Humanos , Hibridização In Situ , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Coloração e Rotulagem , Neoplasias do Colo do Útero/virologia , Displasia do Colo do Útero/virologiaRESUMO
Mammary tumour cytosols prepared for steroid receptor assay showed pH values above or below 7.3-7.5 in 50% of the samples. An increase of 0.6 pH units was observed after the addition of dextran coated charcoal. To test the sensitivity of the assay systems to pH changes, breast cancer cytosols were adjusted to pH between 6.0-9.0 before incubation with ligand at 0 degrees C. ER was then separated by isoelectric focusing in a pH gradient between 9.5-3.5 in a routine assay including a dextran coated charcoal step before focusing. PgR was assayed by electrofocusing as well as by a conventional DCC method and Scatchard plot. Unadjusted control cytosols were run in parallel. At pH above or below 7.0-8.0 recovery of receptor was reduced, the loss being most prominent at low pH. At low pH levels Scatchard plots appeared to be of curvo-linear shape. The effect of low intracellular tumour pH on receptor-ligand affinity in vitro may be of great importance as well in vivo, and should be taken into consideration in the therapeutic situation.
Assuntos
Neoplasias da Mama/metabolismo , Citosol/análise , Receptores de Esteroides/análise , Humanos , Concentração de Íons de Hidrogênio , Focalização IsoelétricaRESUMO
Phosphate and TRIS buffers of low molarity commonly used for steroid receptor assays were studied for their pH stability. Increased temperature from 0-37 degrees C lowers the pH of TRIS buffer, while phosphate buffer remains approximately constant. Addition of dextran and charcoal may change the pH unacceptably, depending on the charcoal quality.
Assuntos
Carvão Vegetal , Dextranos , Concentração de Íons de Hidrogênio , Receptores de Esteroides/análise , Temperatura , Neoplasias da Mama/análise , Soluções Tampão , Reações Falso-Negativas , Reações Falso-Positivas , Feminino , Humanos , Fosfatos , TrometaminaRESUMO
Crude homogenates and cytosols of 31 breast cancer biopsies were analyzed for estrogen receptor, using the ER-EIA monoclonal assay (ABBOTT Laboratories). The recovery of the receptor was significantly higher in all homogenates than in their corresponding cytosols, in six of which the receptor was below the level of detection. The range of the ratio between homogenate and cytosol concentrations was 0.9-8.1 for 25 positive biopsies. If the recent hypothesis of nuclear localization of the receptor is accepted, the values obtained with crude homogenates would more correctly reflect the true concentration of the receptor in the cells.
Assuntos
Neoplasias da Mama/análise , Receptores de Estrogênio/análise , Biópsia , Citosol/análise , Feminino , HumanosRESUMO
False positivity is reported of in situ PCR reactions in a direct incorporation assay with digoxigenin-labelled dUTP. It is recommended that in situ hybridization with specific labelled probe replaces the direct incorporation method for the detection of in situ PCR amplicon.
Assuntos
Hibridização In Situ/métodos , Linfonodos/patologia , Reação em Cadeia da Polimerase/métodos , Apoptose , Biópsia , Células Cultivadas , DNA , DNA Viral/análise , DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxiuracil , Digoxigenina , Reações Falso-Positivas , Fibroblastos/citologia , Herpes Simples/diagnóstico , Humanos , Proteínas Tirosina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2 , Simplexvirus/genética , Simplexvirus/isolamento & purificação , Taq Polimerase , Translocação GenéticaRESUMO
494 tubuloductal breast carcinomas obtained at operation were assayed for ER and PgR with short-term ligand incubation and isoelectric focusing. Plasma FSH and E2 concentrations available from 156 of the patients showed strictly premenopausal endocrine conditions in patients 45 years or younger; strictly postmenopausal conditions were found at 55 years or older. ER concentrations were significantly lower in biopsies from premenopausal compared with those from postmenopausal patients. ER concentrations assayed in intervening perimenopausal age period were not statistically different from the premenopausal period. An arbitrarily chosen cut off level to differentiate receptor low from receptor high tumours divided premenopausal assays into two equal parts; those from postmenopausal patients were cut at the second lower percentile. Arbitrary cut off levels ignorant of menopausal status should be replaced by fractionation of low and high receptor tumours on a percentile or quartile basis. Clinically, subgroups or subpopulations of patients should be identified with regard to endocrine and/or receptor status and evaluated separately.
Assuntos
Neoplasias da Mama/análise , Menopausa , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Adulto , Fatores Etários , Idoso , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Pessoa de Meia-IdadeRESUMO
Germ line point mutations in the RET proto-oncogene have been implicated in four inherited disorders: multiple endocrine neoplasia 2A (MEN 2A) and 2B (MEN 2B); familial medullary thyroid carcinoma (FMTC); and Hirschprung's disease, a congenital lack of enteric plexus neurons. Oncogenically activated RET has also been demonstrated in some sporadic medullary thyroid tumors, which show somatic missense mutations in the same regions as those found in MEN 2B. Upon screening archival sporadic MTC tumor tissue by nonradioactive single-strand conformational polymorphism analysis (SSCP), a markedly divergent exon 11 pattern was found in an unusually aggressive neoplasm. Sequencing of PCR amplified DNA revealed the deletion of nine bases encompassing a key cysteine codon at position 1831-3, often altered in MEN 2A. Normal thyroid tissue from the same patient showed a normal SSCP pattern and sequence for this exon. This novel somatic mutation further implicates the RET proto-oncogene in the development of MTC.
Assuntos
Carcinoma Medular/genética , Cromossomos Humanos Par 10/genética , Proto-Oncogenes/genética , Deleção de Sequência/genética , Neoplasias da Glândula Tireoide/genética , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Proto-Oncogene MasAssuntos
Linfócitos/imunologia , Timo/citologia , Animais , Sítios de Ligação de Anticorpos , Membrana Celular/imunologia , Células Cultivadas , Embrião de Galinha , Concanavalina A/farmacologia , Imunofluorescência , Lectinas/farmacologia , Microscopia Eletrônica , Técnicas de Cultura de Órgãos , Timidina/metabolismo , TrítioAssuntos
Hematopoese , Linfócitos , Timo/fisiologia , Animais , Antígenos , Sangue , Contagem de Células , Divisão Celular , Embrião de Galinha , Concanavalina A/farmacologia , Meios de Cultura , Ativação Linfocitária , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos CBA , Técnicas de Cultura de Órgãos , Oxigênio , Timo/embriologiaAssuntos
Biópsia por Agulha , Linfócitos/patologia , Linfoma/patologia , Sítios de Ligação , Biópsia , Membrana Celular/ultraestrutura , Cromatografia em Gel , Concanavalina A , Fluoresceínas , Humanos , Linfonodos/patologia , Linfonodos/ultraestrutura , Linfoma Difuso de Grandes Células B/patologia , Linfoma não Hodgkin/patologia , Microscopia de Contraste de Fase , Miastenia Gravis/patologia , Tonsila Palatina/citologia , Tonsila Palatina/patologia , Coloração e Rotulagem , Timoma/patologia , Timo/citologia , Timo/patologiaRESUMO
This report demonstrates a previously disregarded source of error in the count of lymphocytes that have been fixed in Turk's dilution fluid (acetic acid and gentian violet). If the dilution fluid does not contain any serum protein or other proteins, a large proporation of the lymphocytes will stick to the walls of the mixing vessel. The use of plastic or siliconized vessels does not prevent this. The number of cells which in that way will be eliminated from the suspension is dependent on several variables, such as the type of mixing procedure and mixing vessel, the compensation of the dilution fluid and the cell density. As a consequence it is difficult to give any correction factor for such a count in a haemacytometer counting chamber. More than 30% of the cells may disappear under generally obtained conditions. This source of error is to some extent also involved in the white blood cell count, despite the presence of about 1% protein in the dilution fluid from the serum and lysed erythrocytes. To prevent this loss of cells either bone serum albumin, to a concentration of 5%, or the detergent Certrimide, to a concentration of 2-5 mM, may be added to the dilution fluid.
Assuntos
Adesão Celular , Contagem de Leucócitos/métodos , Linfócitos , Animais , Humanos , Ratos , Manejo de EspécimesRESUMO
In 30 patients with multiple myeloma who were scheduled for peripheral blood stem-cell transplantation, a quantitative analysis of the stem cells following enrichment by anti-CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele-specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem-cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi-nested PCR combined with a real-time quantitative PCR. In order to accomplish a statistically adequate end-point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34-enriched fraction. The CD34 selection procedure resulted in a median 28-fold enrichment of CD34+ haemopoietic precursor cells. The stem-cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).